Viral infections in the lower respiratory tract cause severe disease and are responsible for a majority of pediatric hospitalizations. Molecular diagnostic assays have revealed that approximately 20% of these patients are infected by more than one viral pathogen. Clinical data indicate that disease severity can be enhanced, reduced or be unaffected by viral co-infection. However, it is not clear how unrelated viruses interact within the context of a complex host to determine disease severity. The long-term goal of this research is to uncover the causal relationships between co-infection and the resulting respiratory disease severity. Variables that will potentially predict disease severity include viral strains, doses, timing, viral competition, genetic variation in the host, and the immune response. The proposed research will develop a murine model with cellular and organismal components and a human in vitro model to test the central hypothesis that respiratory viral co- infections change disease severity both by direct viral interactions and by modulating host responses. Statistical and stochastic modeling will reveal the complex interactions between heterologous viruses within their shared target cells and host organisms. In Aim 1, a mouse strain that exhibits mild, moderate, or severe disease when infected with three respiratory viruses individually will be infected with pairwise combinations of these viruses as concurrent and sequential co-infections. Co-infection variables that lead to differences in morbidity and mortality compared to individual virus infections will be identified. Pathology response variables, including viral loads, inflammatory cells, and histopathology will be analyzed and statistical models will be developed to reveal how both infection variables and pathology response variables predict disease severity during co-infection. Lung transcriptome analysis and complex systems modeling will be used to elucidate mechanisms of host responses that result in differing disease outcomes during co-infection. In Aim 2, viral co- infections will be performed in respiratory epithelial cells in vitro, to determine the effects of co-infection on viral growth dynamics and the response of infected cells. This will reveal the complex interactions between co- infecting viruses within their shared target cells in the respiratory tract. Parallel experiments in murine and human cell lines will test the generality of our findings and may allow us to make predictions about how viral co-infections affect disease severity in humans. Completion of the project aims will result in understanding how interactions between co-infecting viruses, their target cells, and the immune system dictate disease severity during respiratory viral co-infections. Modeling these complex interactions will lead to testable hypotheses about the mechanisms that regulate disease severity during infection by heterologous respiratory viruses.